A thioacetamide-induced liver fibrosis model for pre-clinical studies in microminipig

Abstract

Drug-induced liver fibrosis models are used in normal and immunosuppressed small animals for transplantation and regenerative medicine to improve liver fibrosis. Although large animal models are needed for pre-clinical studies, they are yet to be established owing to drug sensitivity in animal species and difficulty in setting doses. In this study, we evaluated liver fibrosis by administering thioacetamide (TA) to normal microminipig and thymectomized microminipig; 3 times for 1 week (total duration: 8 weeks). The pigs treated with TA showed elevated blood cytokine levels and a continuous liver injury at 8 weeks. RNA-seq of the liver showed increased expression of fibrosis-related genes after TA treatment. Histopathological examination showed degenerative necrosis of hepatocytes around the central vein, and revealed fibrogenesis and hepatocyte proliferation. TA treatment caused CD3-positive T cells and macrophages scattered within the hepatic lobule to congregate near the center of the lobule and increased αSMA-positive cells. Thymectomized pigs showed liver fibrosis similar to that of normal pigs, although the clinical signs tended to be milder. This model is similar to pathogenesis of liver fibrosis reported in other animal models. Therefore, it is expected to contribute to research as a drug discovery and pre-clinical transplantation models.

Figure 1

(A) Scheme depicting TA administration to microminipigs is shown. The TA is withdrawn according to the pig's physical condition. For more information on drug withdrawal, refer to Table 1. (B) Body weight changes at 8 weeks of observation. (C) Elevated cytokine concentrations before and 1 week after administration in TAA and TE-TAA groups were shown as fold change. Cytokines elevated more than twofold were picked up and shown in the graph. (D) Food intake changes during 8 weeks are shown (*: TAA vs. TE-TAA, p < 0.05). (E) Activity changes at pre, 4, and 8 weeks of the experiment are shown. (†: pre vs. 4 weeks, p < 0.05).

Figure 2

(A) Blood biochemical test results. (*: TAA vs TE-TAA) (B) Blood hyaluronic acid at pre, 4, and 8 weeks after injected TA. (C) ICG retention rate at pre, 4, and 8 weeks after injected TA. (†: pre vs. 4 weeks, p < 0.05).

Figure 3

(A) Fluorescence-observed biopsy liver samples are shown using CHP staining. DAPI is represented in blue, and CHP in red. (B) The area of fluorescent regions was calculated as a percentage per tissue area. There is no difference in the percentage of TAA and TE-TAA.

Figure 4

(A) Echographic, macro, and HE images at 8 weeks. (B) The area of the portal and central venous vessel lumen in the liver lobule was evaluated. Dilation of portal venules (PV) and main vein (CV) vessels compared to control vessels. (*: TAA vs TE-TAA) (C) Inflammation scoring of the liver tissue as assessed by an expert pathologist is shown. (D) Immunohistochemical staining images of CD3, Iba1, and αSMA. (E) Percentage of CD3 in the liver lobule is divided into three areas. Area 3 had a higher percentage of CD3-positive cells than the other areas. (*: Area 3 vs Area 1 or Area 2).

Figure 5

Volcano plots of the liver RNA-seq expression analysis results of TAA and TE-TAA compared to that of the control. DEGs used the criteria of |log2 (Fold Change, FC)| > 1 and adjusted p value (padj) < 0.05. (B) Staining images of AZAN, Sirius red, Collagen IV, and Ki67. (C) Fibrosis area calculated from Sirius red staining positive area. (D) Ki67-positive cell rate per 100 hepatocytes expressed as a percentage. (E) Liver fibrosis scoring by pathology experts is shown.